Protease activated receptor-2 contributes to heart failure

Abstract

Heart failure is a major clinical problem worldwide. Previous studies have demonstrated an important role for G protein-coupled receptors, including protease-activated receptors (PARs), in the pathology of heart hypertrophy and failure. Activation of PAR-2 on cardiomyocytes has been shown to induce hypertrophic growth in vitro. PAR-2 also contributes to myocardial infarction and heart remodeling after ischemia/reperfusion injury. In this study, we found that PAR-2 induced hypertrophic growth of cultured rat neonatal cardiomyocytes in a MEK1/2 and p38 dependent manner. In addition, PAR-2 activation on mouse cardiomyocytes increased expression of the pro-fibrotic chemokine MCP-1. Furthermore, cardiomyocyte-specific overexpression of PAR-2 in mice induced heart hypertrophy, cardiac fibrosis, inflammation and heart failure. Finally, in a mouse model of myocardial infarction induced by permanent ligation of the left anterior descending coronary artery, PAR-2 deficiency attenuated heart remodeling and improved heart function independently of its contribution to the size of the initial infarct. Taken together, our data indicate that PAR-2 signaling contributes to the pathogenesis of hypertrophy and heart failure.

Figure 1. Activation of PAR-2 leads to ERK1/2 and p38-dependent cardiomyocyte hypertrophy in vitro.

A: Changes in the total cell area of murine cardiomyocytes were analyzed after 72 hours of stimulation with PAR-2 AP in the presence or absence of MEK1 or p38 inhibitors (9–12 separate wells and 45–60 cardiomyocytes per condition, averaged from two independent cardiomyocyte isolations). B–C: Expression of ANF and BNP in cardiomyocytes 72 hours after PAR-2 AP stimulation in the presence or absence of MEK1 or p38 inhibitors (N = 5–8 per condition). * p<0.05 vs control cells; *** p<0.001 vs control cells; + p<0.05 vs PAR-2 AP treated cells without MAPK inhibitor.

Figure 2. Heart specific PAR-2 overexpression.

Northern blot analysis of PAR-2 mRNA expression in different organs from αMHC-PAR-2 (line 12 and 18) and littermate controls (WT) mice. Overexposed blot demonstrating expression of endogenous PAR-2 mRNA in organs is shown on Figure S2.

Figure 3. Myocardial PAR-2 overexpression leads to cardiac hypertrophy in mice.

A: Representative cross-sections of one year old hearts from WT and αMHC-PAR-2 mice. Sections were stained with hematoxylin and eosin (Bar  = 2.0 mm). B: Expression of pro-hypertrophic gene quantified by real-time PCR in WT (open boxes) and αMHC-PAR-2 (grey filled boxes) at the age of one year (7–8 mice per group). C: The organ weight to body weight ratio was determined in one year old WT (open bars) and αMHC-PAR-2 (grey filled bars) mice (8 to 15 mice per group). * p<0.05.

Figure 4. Cardiomyocyte-specific overexpression of PAR-2 results in inflammation of the heart.

A: mRNA expression of inflammatory mediators in hearts of one year old WT (open boxes) and αMHC-PAR-2 (grey filled boxes) mice (7–8 mice per group). B: Fold changes in MCP-1 and IL-6 (C) protein levels in the culture supernatant from WT (open boxes) and αMHC-PAR-2 (grey filled boxes) mouse cardiomyocytes stimulated with PAR-2 AP for 24 hours. (N = 4–9 for control and N = 9–13 for PAR-2 AP). * p<0.05. ** p<0.05 vs. stimulation with scramble peptide (control).

Figure 5. Cardiomyocyte-specific overexpression of PAR-2 results in heart fibrosis.

A: Representative cross-sections of one year old WT and αMHC-PAR-2 hearts stained with Masson's Trichrome. B–C: mRNA expression of pro-fibrotic genes and MMPs and TIMPs in the heart of one year old WT (open boxes) and αMHC-PAR-2 (grey filled boxes) mice (7–8 mice per group). * p<0.05.

Figure 6. PAR-2 contributes to heart remodeling after permanent LAD occlusion.

A: Cardiac troponin I plasma levels, as marker for cardiac injury, in sham and LAD artery occluded WT (open boxes) and PAR-2^−/− (black boxes) mice 24 hours after surgery (N = 4 for sham and N = 10–13 for LAD). B: Systolic left ventricular internal diameter (LVIDs) before and 4 weeks after occlusion of the LAD artery measured by echocardiography. Changes in heart function calculated as (C) fractional shortening (FS) and (D) ejection fraction (EF) before and after LAD artery occlusion. E: Cardiac hypertrophy shown as ratios of the heart weight to tibia length (HW∶TL) 28 days after permanent LAD artery occlusion. (N = 8–13 per group) F: Representative cross-sections of hearts from PAR-2^+/+ and PAR-2^−/− mice 4 weeks after LAD occlusion stained with Masson's Trichrome. (Bar  = 1.0 mm). * p<0.05 vs. day 0 within the same genotype; # p<0.05.